The invention is mainly related, although not exclusively, with a procedure for a rapid, cost-effective, sensitive and specific quantitation of the accumulative expression of proteins which are usually transiently expressed in the cytoplasmic membrane and reflect a functionally activated specific state in response to a stimuli which has acted in vivo or has been administered in vitro, through the use of compounds able of selectively inhibiting the cleavage and secretion of cytoplasmic membrane proteins to the extracellular media without affecting the functionality of the cell. The invention can be used both with normal and pathological cells present in homogeneous or heterogeneous cell samples for any purpose which requires either the analysis of the expression of membrane proteins associated with the functional activation of the cell or the stabilization of membrane proteins, particularly for diagnostic, prognostic and treatment monitoring purposes.
At present it is well known that any functional process which takes place at the single cell level is reflected, among other manifestations, by the existence of changes in the expression of different cell proteins. Among these modifications, particularly relevant are those changes in the expression of membrane proteins, where they act as receptors and ligands for the transmission of cell-to-cell and cell-extracellular protein signals. Frequently, once a protein has played its role in the cell membrane or, sometimes immediately after a protein has reach ed the cytoplasmic membrane of the cells that have produced it, these proteins are cleaved and leare the cell membrane into the extracellular media through the activity of proteases and especially membrane proteases which specifically act on the cells where they are expressed. Thus, the expression of these membrane proteins frequently occurs in a transient way for variable periods of time, their expression depending not only on their production rate but also on the speed at which they are cleaved from the cell membrane and secreted to the extracellular media.
For a long time it is well known that the potential relevance of the measurement of these membrane proteins as functional activation markers in different cell types, is high. Nevertheless, since their expression on the cell surface is transient, the analysis of their levels on the cytoplasmic membrane has not been routinely used. Alternatively, measurements of these proteins have been made at three different levels: 1) quantitation of the protein secreted to the extracellular media (soluble protein), 2) qualitative evaluation (presence/absence) of the expression of the protein on the cytoplasmic membrane and, 3) the combined assessment of both forms of the protein. However, such determinations do not provide a specific and/or sensitive information about the production of these proteins in individual cells. Because of this, more recently, methods have been developed which allow the quantitation of the overall production of specific proteins for a specific period of time at the single cell level. For that purpose chemical compounds which induce a blockade of the intracellular transport of secretion vesicles such as brefeldin A or monensin have been used to induce the intracellular accumulation of proteins produced in response to a specific stimuli that can occur in vivo or be administered in vitro. The use, in this later group of techniques, of agents that block the transport and extracellular secretion of proteins in a non-specific way at the cytoplasm level has three major disadvantages: 1) their use can be associated with unwanted changes in other cell functions, 2) the use of this methodology would not block the release outside the cell of those molecules of the protein which had reached the cell membrane prior to the administration of the blocking agent and, 3) for the detection of the proteins of interest at the single cell level the use of fixation and permeabilization solutions is required, which decreases the sensitivity of the method for the detection of the protein and represent a limitation for its objective quantitative analysis.
Up to now, no procedure has been described which, with minimum sample manipulation, allows the functional analysis of activation of individual cells, based on the stabilization and both the quantitative and accumulative analysis of the levels of membrane proteins whose expression on the cell surface is usually transient.
Therefore a goal of this invention consists on proposing a solution for the study of the functional activation of leukocytes, platelets and other cells either produced in vivo or induced in vitro, based on the stabilization of proteins from the cytoplasmic membrane and their detection in unmanipulated samples using quantitative cytometric techniques.
In a similar way, another aim of this invention consists of knowing the kinetics of the activity of proteases which participate in processing, cutting and releasing outside the cell membrane, proteins which are associated to functional activation processes from leukocytes, platelets and other cells that can be induced either in vivo or in vitro.
More specifically and in agreement with what has been described above, the procedure of this invention for the study of the functional activation of leukocytes, platelets and other cells, produced either in vivo or induced in vitro, is based on the stabilization on the cytoplasmic membrane of proteins which have a transient expression on the cell surface and their detection using quantitative cytometric techniques, in the absence of additional sample manipulation, being comprised of the steps of:
1) sequential incubation of the sample with an inhibitor (or a combination of inhibitors) specific of the protease or proteases that process these membrane proteins and a mixture of antibodies directly conjugate to fluorochromes.
2) Detection, using quantitative cytometric techniques (for instance, flow cytometry), in a direct way and in the absence of additional sample manipulation, of the expression of the accumulated membrane proteins through the measurement of the fluorochromes directly bound to the cells through the conjugated antibodies.
3) Analysis of the results obtained using a multiparametric analytical approach for the identification of the subsets of cells of interest and to identify within them, those that express the stabilized membrane proteins. quantifying their expression in an objective way based on the fluorescence intensity detected.
After obtaining the sample in the presence of the protease inhibitors mentioned earlier, the use of procedures for the in vitro stimulation of the functional activation of cells, staining with antibodies, the adjustment and calibration of the cytometer are performed in accordance to widely described methods which are recommended for the quantitative analysis of protein expression on the cell membrane. For the selection of the specific cell subsets present in the sample in which it is desired to perform the determination, antibodies will be used that specifically identify those cells and that allow their discrimination from other subtypes of cells present in the sample, therefore avoiding additional sample manipulation.
For the analysis of the results as well as for the accurate and precise quantitation of the expression of each protein of interest, different software programs can be used from which the PAINT-A-GATE PRO(trademark) software is especially useful. During the analysis, apart from the negativity/positivity for each antibody staining one or more specific cell subsets, fluorescenceintensity expressed in objective units including median, mean and dispersion values will be used.
The invention can be used both in normal and in pathologic samples from humans or animals, vegetables, bacterias and other microorganisms, obtained in vivo, stored or treated in vitro, for all the purposes in which the analysis of either the status or the capabilities of functional activation of leukocytes, platelets or other cells is required.
As it can be deduced from what has been described above, the stabilizing compounds (protease inhibitors), the antibodies and/or the fluorochromes can vary mainly depending on the type of proteins functions and/or the types of cells to study, or depending on the type of sample used. Such protease inhibitor is a monoclonal antibody, an aminoacid or aminoacid derived product or a peptide.
In addition, variations in which the number of antibodies used in combination with a fluorochrome is higher than one and those modifications of by the technique in which monoclonal antibodies conjugated with more than one fluorochrome are used, are included in this invention. A pool or more than one pool of monoclonal antibodies conjugated with the saine fluorochrome and directed to one or more different cell subpopulations can be used. Each pool of antibodies used being conjugated with a different fluorochromes.
Finally, in this invention are included those variations in which, after incubating the sample with the antibodies in the presence of stabilizing substances, these can be removed in order to explore the kinetics of action of these proteases that participate in processing, cutting, and releasing outside the cell of membrane proteins associated to the processes of functional activation of leukocytes, platelets and other cells produced in vivo or induced in vitro.
Moreover, in this invention, apart from the staining of the subpopulations of cells and the stabilized proteins under study, the measurement of other surface markers including oncoproteins and proteins related to cell cycle, apoptosis or cell activation and differentiation, can also be performed.
In order to explore such kinetics of action of the proteases involved in processing, cutting and releasing outside the cell those membrane proteins which are associated with the processes of functional activation produced in vivo or induced in vitro of leukocytes, platelets and other cells, after performing the incubation of the sample with the antibodies in the presence of the stabilizing substances, these later can be either removed or their effects reversed.
With this invention we significantly optimize the study of the functional activation of leukocytes, platelets and other cells in biological samples activated in vivo or stimulated in vitro, also allowing the analysis of the kinetics of action of membrane proteases.
It should also be considered that the possibility of stabilizing membrane proteins whose expression in the cell surface is transient or even lasting for very short periods of time provides a detailed information on the functional activation status of the cells analyzed.
The invention can be used for the identification of cells, based on the presence or absence of staining for one or several antibodies or combinations of antibodies used or based on the fluorescence intensity obtained for them.
It should be noted that according to the present invention, the incubation with the membrane protein stabilizing agent could also be performed in vivo prior to obtain the sample.